Multi-functional and configurable assay

ABSTRACT

Novel magnetic assay methods and systems. According to a preferred embodiment, a chromatographic medium is provided that is designed to be contacted with a test solution having activated magnetic particles such that the solution flows bilaterally thereacross. A magnetic field, generated by a magnet or electromagnet, is selectively applied to the medium which causes the charged particles to become substantially bound at a site on the medium specified by the position of the magnet. The assay is multi-configurable and can be modified to isolate target cells and grow cell cultures.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation-in-part of pending U.S.application Ser. No. 11/106,949, filed Apr. 15, 2005, entitledMULTI-FUNCTIONAL AND CONFIGURABLE ASSAY, which is a continuation-in-partof pending U.S. application Ser. No. 10/068,040, filed Feb. 5, 2002,entitled SYSTEMS AND METHODS FOR PERFORMING MAGNETIC CHROMATOGRAPHYASSAYS, and now issued as U.S. Pat. No. 6,991,912 on Jan. 31, 2006,which is a divisional patent application of U.S. patent application Ser.No. 09/668,966, filed Sep. 25, 2000, and now issued as U.S. Pat. No.6,713,271 on Mar. 30, 2004, which was a continuation-in-part of U.S.patent application Ser. No. 09/418,864, filed Oct. 15, 1999, and nowissued as U.S. Pat. No. 6,136,549, issued on Oct. 24, 2000, theteachings of all of which are expressly incorporated herein byreference.

STATEMENT RE: FEDERALLY SPONSORED RESEARCH/DEVELOPMENT

Not Applicable

BACKGROUND

Ligand-receptor assays or immunoassays are well-known in the art. Sincetheir introduction in 1971, such assays have been utilized in a varietyof applications to detect minute amounts of hormones, drugs, antibodies,and other substances suspected of being present in a given fluid sample.In this regard, researchers equipped with enzymes, antibodies, geneprobes, and other reagents have made it possible to create chemicaldetection schemes for almost any. compound of interest in a greatdiversity of applications. Among these applications are: commercialproduction of pharmaceuticals and food stuffs; food safety; diagnosisand treatment of disease in medical, veterinary, and agriculturalenvirons; and detection and eradication of toxins in the environment.Common to all such applications is the requirement that chemicaldetection be performed in a timely, reliable, and cost effective manner.

Generally, bioassay schemes are developed and commercialized in formatssuitable for use in laboratories equipped with general purposeinstrumentation. Examples of these formats include immunoassay and DNAhybridization performed in test tubes, cuvettes, microtiter plates,columns, and electrophoretic gels. These formats usually includeelaborate operational procedures and require frequent calibration usingseveral calibrants which contain the analyte of interest at differentconcentrations. As a consequence, the high cost and complexity ofoperation associated with such formats limits widespread utilizationthereof.

To address such drawbacks, developers and end users of immunoassays areincreasingly replacing conventional bioassay formats which use testtubes, cuvettes, microtiter plates, columns, and electrophoretic gelswith thin film chromatographic devices known as test strips. As is knownin the art, the majority of test strips used for immunochemicaldetection of compounds are so called lateral flow test strips in whichsample and reagents flow within the plane of the test strip.Advantageously, assays configured in a test strip format can producerapid results, are simpler to operate, and are more cost-effective thanconventional formats. Additionally, such test strip assays may beutilized by unskilled laborers and can produce results on-site (i.e.,outside a laboratory facility).

Generally, such assays rely on the binding of analytes by receptors todetermine the concentration of such analytes in a given sample and aretypically characterized as either competitive or non-competitive.Non-competitive assays generally utilize receptors in substantial excessover the concentration of analytes to be determined in the assay.Typical of such non-competitive immunoassays include sandwich assays,which detect the presence of an analyte by binding two receptorsthereto. In such arrangement, the first receptor, which is typically anantibody is bound to a solid phase such that when the analyte ispresent, such analyte becomes affixed thereto. A second receptor havinga label covalently attached thereto, which may comprise a radioactive,fluorescent, enzymatic, dye or other detectable moiety (collectivelyreferred to as tracers), is introduced to the assay which consequentlybinds to the bound ligand, to the extent the ligand is present, andthereafter produces a signal consistent with the presence of suchligand. If the sample does not contain the molecules of interest, thelabeled receptor is carried past the immobilized receptor withoutreacting which, as a consequence, will not cause a change in themembrane. Such non-competitive immunoassays are primarily useful for thedetection of large molecules such as proteins, large hormones ormolecules which have multiple binding sites, such as human chorionicgonadotropin (HCG) and typically will not work with small molecules thathave only one binding site.

Competitive assays, in contrast, generally involve competition between aligand present in a given sample, and a ligand analog having atracer/label covalently linked thereto to permit detection for a limitednumber of binding sites provided by the ligand receptor, which typicallycomprises an antibody bound to a solid phase. Such assays areparticularly suited to detect smaller molecules, such as drugs and drugmetabolites. In this context, drug analogs are utilized that have beencovalently bound to a protein which is then immobilized on a membrane.Antibody specific to the drug is then labeled and immobilized on aporous pad. When a sample is added which is suspected of containing agiven analyte, such sample dissolves the labeled antibody and carries itinto contact with the immobilized drug-protein region. If there islittle or no drug in the sample, a large amount of the labeled antibodyis bound to the immobilized drug-protein region which, consequently,produces a detectable signal. If the sample contains a high amount ofdrug, little or no labeled antibody is bound to the immobilizeddrug-protein region and thus in turn gives little or no signal.

Today, rapid immunoassays generally consists of an adhesive-coveredplastic backing onto which several porous pads and a piece ofprotein-binding membrane are attached. The membrane typically contains asection that has been impregnated with a binding partner (i.e., areceptor or ligand analog). A second pad is typically provided whichcontains a labeled target molecule or labeled antibody protein-bindingmembrane. When a sample suspected of containing a target ligand iscontacted with the immunoassay, such sample dissolves the labeledelement or tracer and the capillary action of the protein-bindingmembrane subsequently draws the sample with tracer dissolved thereininto contact with the impregnated binding partner. When this reactionoccurs, there is a change in the appearance of the binding membrane,with the difference providing a qualitative indication of the presenceor absence of the ligand suspected of being present in such sample.

Typical examples of this form of test strip are those which visuallydisplay two parallel lines (known as capture lines) on a test membrane.Capture lines consist of immobilized capture reagents or receptors whichare preapplied to the test membrane during its manufacture. In thisregard, both virtually all prior art assays, whether competitive ornon-competitive, typically deploy a receptor immobilized on a membrane,as assessed above. A schematic representation of the construction of atypical lateral flow test strip is as follows:

reagent pad//test membrane/capture line/test membrane/capture line/testmembrane//absorbent pad.

where:

symbol/designates a phase boundary within a single chromatographicmedium; and

symbol//designates a union of two separate mediums (chromatographic orother medium).

One of the two capture lines serves as an indication that the test stripperformance has not been compromised. In this regard, such capture lineserves an important function by providing quality assurance andintegrity of the assay, which is generally considered necessary insofaras individual test strip performance can vary greatly. The second ofsuch capture lines becomes visible only when the sample contains anamount of analyte in excess of a minimum concentration (thresholdconcentration). Exemplary of such prior art systems and methodologiesinclude the immunoassay systems and test strips disclosed in U.S. Pat.No. 5,658,723, issued on Aug. 19, 1997, to Oberhardt entitledIMMUNOASSAY SYSTEM USING FORCED CONVECTION CURRENTS and U.S. Pat. No.5,712,170, issued on Jan. 27, 1998, to Kouvonen, et al. entitled TESTSTRIP, ITS PRODUCTION AND USE, the teachings of each of which areexpressly incorporated herein by reference.

Unfortunately, despite their cost-effectiveness and simplicity of use,typical test strip format assays are less accurate, less precise, andless sensitive to analyte presence than conventional formats. As aresult of such drawbacks, the application of test strip format assayshas been limited to semi-quantitative or qualitative assays. Among themore significant factors that contribute to the inaccuracy andimprecision of test strip format assays include the manufacture and useof capture lines. As is widely recognized, the manufacture ofconsistently uniform capture lines requires elaborate material controland manufacturing processes with rigid specifications that must operatewithin narrow tolerances. Moreover, to function properly, most teststrip formats require that the analytes to be detected must be uniformlycaptured in a precise geometry at a precise location on the test stripand that factors such as the ambient humidity present at the time oftest strip manufacture, type of membrane utilized in such manufacturingprocess, and a capture reagent-receptor itself contributing greatly toassay inaccuracies and false readings. A detailed discussion regardingthe drawbacks associated with the binding of protein capture reagents inimmunochromatographic assays can be found in Jones, Kevin D.,“Troubleshooting Protein Binding in Nitrocellulose Membranes”, Part I,IVD Technology, Volume V, No. II, March-April 1999, pages 32-41 and PartII, IVD Technology, Volume V, No. III, May-June 1999, pages 26-35, theteachings of which are expressly incorporated herein by reference.

Of further significant disadvantage is the fact that virtually all teststrip format assays are formed to have a sequential, generally-linearconfiguration so as to facilitate the necessary lateral flowthereacross. Due to the fact that such fluid sample must necessarilymigrate from its starting point across the reagent pad, the testmembrane, and ultimately across the capture line(s) for detecting thepresence of the suspect analyte, a substantial portion of the targetanalyte is often caused to become dispersed or otherwise inhibited fromreaching the bound receptors forming the capture line. As such, asubstantial portion of the target analyte sought to be detected can andfrequently is missed altogether which can adversely affect thequantitative and qualitative results generated by such assays.

Such potential to inadvertently fail to detect the presence of a targetanalyte, whether it be through losing the target analyte sought to bedetected or simply overlooking its presence is particularly problematicwhen attempting to detect the presence of cancer cells in a given fluidsample. For example, in a single 10 ml tube of blood, there areapproximately fifty billion cells, and the presence of so much as onecancer cell among this cell population can be indicative of the presenceof micrometastasis. Utilizing conventional screening techniques, suchblood samples are typically processed to isolate the leukocytes presentin such sample, which advantageously reduces such fluid sample forexample, from 10 ml to between 1.0 to 0.5 ml, which consequently reducesthe cell population from approximately fifty billion to approximatelyone hundred twenty million. Such procedure, however, typically resultsin the loss of cancer cells and as such may inadvertently remove thecancer cells sought to be detected.

In order to screen for cancer cells, such resultant sample is thenportioned and applied to many microscope slides at a ratio ofapproximately one million cells to each slide, via well-known proceduressuch as cytospin. As is known, each slide prepared represents anotherinadvertent loss of cancer cells. Each respective one of the slides mustthen be meticulously scanned using a microscope. In this respect, eachslide is typically divided into four hundred magnified fields comprisedof one square millimeter areas that are each reviewed. Typically, suchscanning process takes on average a half hour per slide. As a result, tothoroughly examine the condensed population of a one hundred twentymillion leukocytes present in one 10 ml blood sample results requiresthe examination of one hundred twenty slides, producing forty eightthousand images that must be examined over a sixty hour period.Accordingly, even to the extent prior art assay techniques are effectiveat labeling a target cell sought to be detected, the process by whichsuch labeled cell is ultimately isolated and detected is inherentlyunreliable, tedious and time consuming.

It is therefore desirable to devise an alternative lateral flow devicewhich can capture analyte at a precise location, and preferably at thestarting point or point of contact at which the fluid sample isdeposited. It would be likewise desirable to devise an alternative assaythat can capture an analyte at such starting point or point of contactin a precise geometry without the use of preapplied capture lines. Thereis also a need for an assay that has greater sensitivity inreproducibility than prior art assays and methods and is likewiseinexpensive, less labor intensive, relatively easy to manufacture, andcapable of being utilized for a wide variety of applications. There isfurther a need for an essay that may capture prepared cell specimensdirectly onto microscopic slides and significantly reduce the time andnumber of required images to be produced therefrom, particularly withrespect to the isolation and detection of cancer cells.

In addition to the foregoing, there is a substantial need in the art foran alternative assay that can be constructed in a variety ofconfigurations to substantially enhance the ability of the assay toperform a specific application. In this regard, it would beexceptionally advantageous for an assay system that is operative toserve as a separations platform for use in not only isolating proteinsand genes, but also performing such fanctions as isolation, sorting,interrogation, and subsequent expansion of propagation of live cells.Such multi-configurable assays would further advantageously be operativeto handle small quantities of compounds and conserve sample volume, tothus conserve the consumption of rare materials, and further beoperative to eliminate, where applicable, conventional ancillaryseparation componentry, such as microtiter plates, separation columns,and the like that can further easily be integrated with many existingimage analysis systems. Still further, there is a need for such a systemthat is of relative low cost, easy to manufacture, and used asmulti-component construction to thus enable the assay to be readilyconstructed or fashioned to optimally perform a specific type ofseparation procedure.

BRIEF SUMMARY

The present invention specifically addresses and alleviates to theabove-identified deficiencies in the art. In this regard, the presentinvention pertains to several novel bioassay methodologies,chromatographic devices, and an optional multimode photometer/analyzerwhich together can perform bioassays with accuracy and precision likethat of conventional laboratory formats while retaining the operationalsimplicity, rapid analysis, and cost-effectiveness like that of teststrip formats. The chromatographic devices and novel bioassaymethodologies of the present invention further minimize problemsassociated with the manufacture of test strips which incorporatepreapplied capture lines and further, can enable an analyte to bedetected in a fluid sample in a manner that efficiently conserves andisolates the analyte present in such sample. Moreover, multimodephotometers, novel test strip devices, and unique chemical analysismethods of the present invention represent a versatile, cost effective,simple, and accurate system which can quantify the amount of a chemicalsubstance present in a sample that has not heretofore been available viaprior art bioassay test strips.

According to a first aspect of the present invention, there is provideda novel magnetic chromatography method which consists of the steps ofcontacting activated magnetic particles suspended in a reaction mixturewith a chromatographic medium (e.g., test strip or chromatographicplate), and thereafter applying a magnetic field thereto. As theactivated magnetic particles flow laterally within the plane of themedium they encounter the applied magnetic field. The applied magneticfield attracts the magnetic particles forming a magnetic barrier thatselectively retains magnetic particles while allowing the reactionmixture to continue to flow laterally thereacross. In an embodimentparticularly useful in performing cell capture assays, the reactionmixture with magnetic particles is contacted upon an intermediateportion of a chromatographic medium having a magnetic field applied atthe point of contact. The reaction mixture is thus caused to flowbilaterally across the medium, with the magnetic particles having theanalyte of interest complexed therewith being captured at the startingpoint, or point of sample introduction, thus conserving the amount ofanalyte in the reaction mixture that would otherwise become lost throughreaction mixture flow.

As such, there is thus eliminated the conventional capture lines formedby bound receptors that are utilized in prior art immunoassays as wellas analyte loss that can occur during the assay. In this regard, acapture line is in effect assembled during the assay, and preferably atthe outset. Advantageously, the magnetic chromatography assay methods ofthe present invention allow test strips and the like to be manufacturedwithout preapplied capture lines. However, the methods of the presentinvention also anticipate a magnetic chromatography test strip havingboth preapplied capture lines and capture lines formed during thebioassay using magnetic chromatography as may be desired for a specificapplication.

The novel methods of the present invention may further deploy one ormore applied magnetic field source(s) applied to the chromatography teststrip assembly to detect multiple spectrophotometric analysis. Forexample, a common bar magnet or magnetic strip can be attached to thetest strip backing with adhesive at one or more locations. Alternately,the magnetic source can be external to the test strip assembly wherebythe magnetic source is selectively positioned in close proximity withthe test strip while magnetic particles flow laterally therewithin. Inpreferred embodiments of the present invention, the source of theapplied magnetic field may comprise either permanent magnets orelectromagnets.

The present invention further includes a novel magnetic chromatographytest strip for performing a bioassay that conserves the amount ofanalyte sought to be detected in a reaction mixture by forming a captureline or zone at the point at which the reaction mixture is contactedwith the test strip. According to a preferred embodiment, the test stripcomprises an elongate backing having first and second ends and anintermediate portion disposed therebetween. Upon the intermediateportion are a first vertical mesh for facilitating vertical flow andsecond lateral mesh for facilitation lateral flow, the latter beingdisposed underneath the first mesh. On opposed sides of the lateral flowmesh are absorbent pads that, in use, cause the reaction mixtureultimately passing through the first and second meshes to flowbilaterally. Such strip further preferably includes at least one magnetthat is positioned underneath the vertical flow mesh and second lateralflow mesh.

In use, a reaction mixture is sequentially caused to flow from thevertical flow mesh, to the lateral flow mesh, and ultimately to thefirst and second absorbent pads disposed on the opposed sides of thelateral flow mesh. Preferably, such reaction mixture is deposited uponthe strip by means of a sample well. The applied magnetic field providedby the magnet attracts the magnetic particles having the analyte ofinterest complexed thereto, which thus causes the same to be selectivelyretained at the site of deposition while the remainder of the reactionmixture continues to flow thereacross and ultimately to the respectiveof the absorbent pads. Such test strips are particularly well suited forthe detection and isolation of cells, such as cancer cells, bacteria andthe like, which are otherwise difficult to identify and isolate throughprior art methods.

To further accomplish the objectives of the present invention, as wellas to enable the novel magnetic chromatography methods to be utilized ina wide variety of applications extending across genomics, microgenomics,proteomics, and cell isolation for use in target identification (e.g.organelle sorting, protein fractionation, or target validation e.g.protein isolation, immune assays, molecular analysis) there is furtherprovided a multi-configurable assay system. According to a preferredembodiment, such multi-configurable assay systems comprise a housingmountable upon a base, the housing defining an interior and having asample well formed thereon into which a reaction mixture containingmagnetic particles, along with any applicable reagents, are deposited.In a preferred embodiment, formed upon the underside of the housingabout the exit opening of the sample well, there is provided atexturized surface, which may comprise a pattern of uniformly arrangedpillars, members, channels and the like that are operative to introduceimpedance of flow of a sample flowing thereacross. In this regard, suchtexturized surface is operative to restrict or impede a sample flow soas to enable an analyte of interest, and in particular, a target cell,to remain more readily confined at a selectively chosen locus uponapplication of a magnetic field, discussed more fully below.

Optionally, absorbent pads will be disposed within the housing onopposed sides of the sample well to enhance bilateral flow of a reactionmixture deposited in the sample well. A base is provided that may beformed to be either permanently or detachably fastened beneath thehousing for receiving the reaction mixture. The base will define atarget area that is aligned with sample well so that substantially allthe reaction mixture is concentrated upon a single locus, and maycomprise a conventional slide or culture/Petri dish, the latter beingoptionally provided with a desired medium to facilitate the growth of acell culture.

A magnetic source, which may be either separate from or attached to thebase, is provided that is aligned with the target area and sample welland operative to apply a magnetic field thereto to thus attract andretain the magnetic particles present in the reaction mixture at thetarget area while allowing the remaining reaction mixture to flowlaterally across the target area. As a consequence, the magneticparticles present in the reaction mixture that have a target analyte(e.g. cell, protein, gene) complexed therewith will thus be captured andretained at the point of introduction, which thus optimally minimizespotential migration of the target analyte.

In refinements of such assay, the base member will comprise aconventional slide that, once the analyte of interest is capturedthereupon, can be viewed, handled and stored per conventional slidepreparations. Alternatively, the base may have a mesh with or withoutgel preparation deposited upon the target area thereof for use in avariety of applications, such as initiating the growth of a cell cultureor serving as a means to suspend receptors that will selectively bind totarget analytes of interest. Additionally such gel preparation may beprovided with other types of reagents and enzymes, such as digestiveenzymes, to selectively cleave proteins or otherwise serve to facilitatea desired biochemical reaction. Per the aforementioned embodiments, thebase may further be provided with one or more layers of mesh to thusfacilitate the direction of the flow of the reaction mixture as well asenhance the ability of the analyte of interest to be captured at thetarget area. Still further, the base may comprise a conventional Petrior culture dish to facilitate the growth of a cell culture. Along theselines, it is contemplated that virtually any type of conventionalsubstrate, and in particular a substrate such as a conventional Petridish that can be used to further promote the growth and examination ofcells to be conducted.

In a further refinement of the invention, the base member may comprise anovel texturized surface that is operative to impede or restrict thesample flow deposited thereon to thus enable an analyte of interest, andin particular a cell, to be more discreetly retained at a selectivelychosen locus. To that end, such texturized surface may comprise aplurality of flow-impeding objects, such as pillars, cylinders,channels, and the like, that are operative to define a physical barrieracross which a sample must flow. By impeding sample flow, the fluidsample and all objects dispersed therein are caused to encounter greaterflow restriction (for example, a greater number of collisions, increasedflow path routes, and the like) that in turn decreases the rate of flowflowing away from the sample well and enables a prolonged amount of timefor the magnetic forces to more thoroughly act upon and retain thetarget analyte of interest at a predetermined locus, typically the pointof sample deposition. According to a preferred embodiment, thetexturized surface may comprise a uniformly distributed pattern ofpillars or cylindrically-shaped members arranged in a generallygrid-like fashion. In one particular arrangement that is presentlyconsidered ideal for facilitating the isolation of cells, such pillarstructure are preferably formed to have a cylindrical shape with a widthof approximately 20 microns and spaced apart approximately 50 micronsfrom one another. Such texturized surface can be formed upon anysuitable substrate upon which a sample is deposited, including but notlimited to conventional slides and the like.

By virtue of its ability to be multi-configurable, the assays of thepresent invention are operative to be utilized in any of a variety ofisolation, sorting and interrogation applications. Exemplary of suchapplications include single slide bench, bench top use, small batchtesting, automated batch processing and continuous high throughputscreening. Such multi-configurable assay, by virtue of its versatility,is exceptionally effective in capturing and isolating target analytes,such as cells, proteins and genes, functionally and structurally intact, even if the same are membrane bound, fragile, or of exceptionallysmall or large size. Likewise, the multi-conformable assays can be usedfor cell engineering and interrogation, cell engineering and expansionor propagation, cell resistance and interrogation and cell sorting andserial interrogation applications. Advantageously, in all suchconfigurations, the assay systems of the present invention will remainclosed systems which thus eliminate many of the handling steps oftraditional methods as well as permits the rapid and easy handling ofspecimens with minimal risk of material exposure to workers and/orcontamination of the sample being processed.

There is further provided as part of the present invention a novelanalyzer comprised of a multimode photometer which can measure frontsurface fluorescence, luminescence and reflectance at a single focalpoint on the test strips of the present invention. According to apreferred embodiment, the multimode photometer consists of a base andoptical canopy which collectively define an optical tunnel into which atleast one test strip may be disposed. The chamber may include a magneticsource or be designed to be placed in close proximity to a magneticsource such that the test strip having activated magnetic particlesflowing laterally therewithin may be caused to become substantiallybound at a specific site or sites upon the test strip. When so arranged,a light or radiation source may be focused upon the test strip disposedwithin the optical tunnel such that the light or radiation may bealigned with the magnetic source and the reflected or emitted light fromthe test strip analyzed for analyte presence. Light and radiation ofdiffering wave lengths may be utilized to determine the presence ofappropriate analytes as per conventional spectrophotometric analysis.Optical filters and photodetectors may further be deployed as may benecessary for a particular spectrophotometric application.

It is therefore an object of the present invention to provide a novelmagnetic chromatography assay and method utilizing a test strip formatthat has greater sensitivity and reproducibility than prior art teststrip assays.

Another object of the present invention is to provide a novel magneticchromatography assay and method that utilizes a test strip format, butdispenses with a need to form a capture line by binding receptors to atest membrane.

Another object of the present invention is to provide a novel magneticchromatography assay and method that can be arrayed in a test stripformat and utilized to provide quantitative analysis.

Another object of the present invention is to provide a novel magneticchromatography assay and method that can form a capture line at thepoint of contact where a reaction member comes into contact with suchtest strip.

Another object of the present invention is to provide a novel magneticchromatography assay and method that can conserve the amount of analytepresent in a reaction mixture by identifying and isolating the same atthe initiation of the performance of such assay.

Another object of the present invention is to provide a novel magneticchromatography assay and method that may be adapted to providequantitative and qualitative analysis for multiple analytes.

Another object of the present invention is to provide a novel magneticchromatography assay and method that is easy to use, of simpleconstruction, and inexpensive to manufacture.

Another object of the present invention is to provide a novel magneticchromatography assay and method that may be utilized to providespectrophotometric analysis, including but not limited to, surfacereflectance, surface fluorescence, and surface luminescence.

Another object of the present invention is to provide a novel magneticchromatography assay and method which may be configured to isolatetarget cells and facilitate the ability to detect the same.

Another object of the present invention is to provide a novel magneticchromatography assay and method which may be adapted to capture cellsdirectly onto slides and culture dishes and facilitate microscopicexamination thereof.

Another object of the present invention is to provide a novel magneticchromatography assay and method which may be configured to performindividual sample analysis, batch sample analysis, and linear-arrayanalysis.

Another object of the present invention is to provide a novel magneticchromatography assay system that is multi-configurable and capable ofbeing utilized in a wide variety of applications.

Another object of the present invention is to provide a novel magneticchromatography assay system that can permit efficient handling of smallor large numbers of specimens, and is readily scaleable from singleslide bench top applications to a continuous high throughput screeningenvironment.

Another object of the present invention is to provide a novel magneticchromatography assay system that is operative to rapidly isolate andsort sub-populations of cells with specific antigenic expression, andcan further optionally be useful in facilitating cellexpansion/propagation.

Another object of the present invention is to provide a novel magneticchromatography assay system that is readily configurable to performassays related to cell isolation, creation of a cell culture, cellengineering, cell resistance and cell sorting and serial interrogation.

Another object of the present invention is to provide a novel magneticchromatography assay system that is configurable to facilitate theisolation and creation of a cell culture that is particularly wellsuited with rare and/or fragile cells.

Another object of the present invention is to provide a novel magneticchromatography assay system that is particularly effective in isolatingcells of interest contained within a sample that facilitates the sampleand all other non-components of interest to more readily flow away fromthe target cells of interest to thus enable the latter to be morereadily isolated and, optionally cultured to create a cell culture.

Another object of the present invention is to provide a novel magneticchromatography assay system that can substantially reduce, if noteliminate, reliance on microtiter plates, separation columns and theneed to perform cell transfer procedures while at the same timeachieving greater cellular capture and minimizing cell losses.

Another object of the present invention is to provide a novel magneticchromatography assay system that eliminates many of the handling stepsof traditional assay methods and further permits the rapid and easyhandling of specimens with minimal risk of bacterial exposure to workersand/or contamination of the sample being processed.

Another object of the present invention is to provide a novel magneticchromatography assay system that provides structure for performingpost-assay assessment and manipulation, including but not limited tocellular testing, lysing and/or protein/organelle isolation andseparation.

Another object of the present invention is to provide a novel magneticchromatography assay and method wherein such assay may be configured tobe reusable or disposable.

Another object of the present invention is to provide a novel magneticchromatography assay and method which will accommodate conventionalreagents prepackaged in unit doses.

Another object of the present invention is to provide a novel magneticchromatography assay and method that can be used for quantitative,semi-quantitative, and qualitative immunoassay of analytes and DNAhybridization assays.

Another object of the present invention is to provide an opticalanalyzer consisting of a multimode photometer for performingspectrophotometric analysis, including but not limited to, surfacereflectance, surface fluorescence, and surface luminescence.

Another object of the present invention is to provide an analyzerconsisting of a multimode photometer which is of simple construction,easy to utilize, and may be configured to perform individual sampleanalysis, batch sample analysis, and linear-array analysis.

Another object of the present invention is to provide an analyzerconsisting of a multimode photometer that, when utilized in conjunctionwith the magnetic chromatography assays of the present invention, may beutilized to quantify the amount of a given analyte at a fixed locationon a test strip assay, irrespective of orientation of such assay andlateral flow of reaction mixture utilized therewith.

BRIEF DESCRIPTION OF THE DRAWINGS

These as well as other features of the present invention, will becomemore apparent upon reference to the drawings wherein:

FIG. 1 a is a perspective view of an assay test strip for using thepractice of the methods of the present invention, said test strip beingconstructed in accordance to a first preferred embodiment.

FIG. 1 b is an exploded view of the components comprising the assay teststrip depicted in FIG. 1 a.

FIG. 1 c is a side view of the assay depicted in FIG. 1 a.

FIG. 2 a is an exploded perspective view of an assay test stripconstructed in accordance with a second preferred embodiment of thepresent invention.

FIG. 2 b is a side view of the assay test strip depicted in FIG. 2 a.

FIG. 3 a is a cross-sectional view of a multimode photometer constructedin accordance with a preferred embodiment, as utilized in the practiceof the methods of the present invention.

FIG. 3 b is a cross-sectional view and block diagram of the multimodephotometer depicted in FIG. 3.

FIG. 4 a is perspective view of the multimode photometer depicted inFIG. 3.

FIG. 4 b is a top view of the multimode photometer depicted in FIG. 3.

FIG. 4 c is an exploded cross-sectional view of the componentscomprising the multimode photometer depicted in FIG. 3.

FIG. 5 a is a top view of a multiplicity of test strips arrayed inparallel rows on a common backing for use in detecting the presence andquantity of one or more analytes from a plurality of samples.

FIG. 5 b is a top view of a multiplicity of test strips utilizing asingle-common absorbent pad having fluid contact with a multiplicity oftest membranes, the latter being arranged in a generally linear fashion.

FIG. 6 a is a top view of an assay test strip constructed in accordancewith a third preferred embodiment of the present invention.

FIG. 6 b is a side view of the test strip depicted in FIG. 6 a.

FIG. 7 a is a top view of an assay test strip constructed in accordancewith a fourth preferred embodiment of the present invention.

FIG. 7 b is a side view of the assay test strip depicted in FIG. 7 a.

FIG. 8 is an elevated perspective transparent view of amulti-conformable assay system constructed in accordance with apreferred embodiment of the present invention.

FIG. 9 is an exploded view of an assay constructed in accordance withthe principles of the assay depicted in FIG. 8.

FIG. 10 is an expanded view of a gel layer deposited upon the mesh ofthe assay depicted in FIG. 9 for use in facilitating the capture,isolation and further biochemical processing of a target analyte presenttherewith.

FIG. 11 is a perspective view of a multi-conformable assay systemconstructed in accordance with a preferred embodiment of the presentinvention as positioned within a Petri dish to facilitate the creationof a cell culture.

FIG. 12 is a partial cross-sectional view of the multi-conformable assayof FIG. 11 having a liquid sample applied thereto.

FIG. 13 is a cross-sectional view of the housing of the assay of FIGS.11 and 12 showing the flow of a sample moving therethrough.

FIG. 14 is a cross-sectional view of the multi-conformable assay andPetri dish depicted in FIGS. 11-13, with the housing of themulti-conformable assay being removed from the Petri dish with thetarget cells of interest remaining within the Petri dish as a locus asdefined at a point whereby magnetic particles bound to the target cellsof interest were drawn into position at the point of deposition by amagnet positioned beneath the Petri dish.

FIG. 15 is a bottom view of an assay housing for use in facilitating theisolation of target cells constructed in accordance with the preferredembodiment of the present invention.

FIG. 16 is a frontal view of the texturized surface formed upon theunderside of the housing depicted in FIG. 15.

FIG. 17 is a perspective view of a slide having a texturized surfacethereon, the latter being operative to restrict a flow of a fluid samplemoving thereacross.

FIG. 18 is a sectional view of the texturized surface depicted in FIG.17.

FIG. 19 is a cross-sectional view of a texturized surface depicted inFIG. 18.

DETAILED DESCRIPTION

The following detailed description and the accompanying drawings areprovided for the purpose of describing certain presently preferredembodiments of the invention only, and are not intended to limit thescope of the claimed invention in any way. In this regard, there isdisclosed herein a novel assay system and method that, unlike prior artassay systems, and in particular test strip assays, can quantitativelyand qualitatively detect the presence of an analyte, control,calibrator, or combination thereof in a given fluid sample withextraordinary precision and reproducibility. Moreover, the novel assaysand methods of the present invention provide all of the advantagesassociated with conventional test strips assays insofar as the same neednot undergo remote analysis at a laboratory facility and further, do notrequire handling by trained professionals. There is further provided anovel analyzer, which comprises a multimode photometer, is useful inconducting spectrophotometric analysis in conjunction with the assaysand methods of the present invention.

Referring now to the drawings, initially to FIGS. 1 a-1 c, there isshown a preferred embodiment of a test strip for use in magneticchromatography. The test strip is comprised of a test membrane 1 havinga reagent zone 2 at its one end and an absorbent pad 3 at its other end.These components are attached to a backing 4 made. of plastic, glass orother suitably rigid material. Similar to prior art test strips, thetest strip is simple to manufacture by lamination.

Another embodiment of the test strip for use in the practice of thepresent invention is shown in FIGS. 2A and 2B. In this embodiment of theinvention, there is provided a reagent pad 5 at one end and absorbentpad 3 at the respective other end. In this regard, the reagent pad 5 isshown partially overlapping the test membrane 1 to thus produce agreater degree of saturation thereacross, as may be desired for a givenapplication.

In either of the test strip embodiments depicted in FIGS. 1 a-1 c andFIGS. 2 a-2 b, it will be readily understood and appreciated by thoseskilled in the art that the same are designed to produce a lateral flowor path of migration that extends from the reagent pad 5 to theabsorbent pad 3 at the other end. As per conventional test strip assays,the lateral flow of a reaction mixture across the test membrane 1provides a basis for conducting chemical analyses over a given surfacearea (i.e., the test membrane 1).

Unlike prior art test strip assays, however, the assays and methods ofthe present invention do not utilize a capture barrier formed by boundreceptors formed along a portion of the test membrane 1, but ratherutilize a novel magnetic approach to generate such capture lines. Inthis regard, due to the novel methods and systems by which capture linesare generated via the present invention, it will be recognized thatalthough the test strip configurations depicted in FIGS. 1 and 2 may bereadily utilized in the practice of the present invention, the onlyessential element thereof comprises a chromatographic medium, such as atest strip or chromatographic plate, upon which a test sample may flowlaterally thereacross. Accordingly, it will be understood that a path ofmigration need not necessarily be formed, as per conventional teststrips and the like, in order to practice the present invention.

Test Membrane

The test membrane 1 can be selected from any available material havingappropriate thickness, pore size, lateral flow rate, and color. It ispreferred that the test membrane be made from a material which has a lowaffinity for the analyte and test reagents. This is to minimize or avoidpretreatment of the test membrane to prevent non-specific binding ofanalyte and/or reagent. Polyester is an example of a suitable testmembrane material.

Reagent Pad

The (optional) reagent pad 5 can contain all or a portion of thereagents necessary to complete the assay. Reagents can include a captureligand and reporter ligand which specifically bind different regions ofthe analyte to be detected in a given sample. The capture ligand can becovalently bound or absorbed to the surface of magnetic particles.Capture ligands can also be bound indirectly using binding partners suchas anti-IgG antibody, streptavidin/biotin, and others. The reporterligand is covalently bound to a dye, particle, radioisotope, or enzymewhich produces fluorescence or luminescence. The reagent pad 5 can alsocontain stabilizers, buffers, surfactants and other agents which improvethe performance of the assay. The reagent pad 5 receives the sample andall subsequent liquid reagents used to perform the assay. The reagentpad 5 also can be selected from any available material havingappropriate thickness, pore size, and flow rate. It is preferred thatthe reagent pad be made from a material which has a low affinity for theanalyte and test reagents. Again, this is to minimize or avoidpretreatment of the reagent pad 5 to prevent non-specific binding ofanalyte and/or reagent. Polyester and porous polyethylene are examplesof suitable reagent pad 5 materials. The reagent pad 5 should be ofsufficient size and void volume to accept the entire sample volume.

In some embodiments of the invention the reagent pad 5 may not be aphysically separate component. Rather, instead the reagents can bestored in a reagent zone 2 formed on the test membrane 1 itself. Inother embodiments of the invention, the reagent pad 5 does not containreagents and instead is used as a liquid reagent receiving pad. As willbe appreciated by those skilled in the art, by forming such reagentzones upon the test membrane as a substitute for reagent pads, the costand complexity of manufacturing is substantially reduced insofar as thereagent pad component may be eliminated altogether. In this regard, thenon-binding properties of the test membrane, coupled with the ability toform a capture line magnetically, as discussed more fully below,eliminates the need to design a test strip whereby a fluid sample mustnecessarily flow sequentially in one direction so that a given fluidsample with reagents thoroughly and precisely comes into contact with aconventional capture zone defined by a multiplicity of bound antibodies.

Absorbent Pad

The (optional) absorbent pad 3 should have absorbent capacity sufficientto contain all liquid volumes used during the test procedure. Cottonfiber and absorbent paper are examples of suitable absorbent pad 3materials. As discussed above, however, the absorbent pad is optionalinsofar as the chromatographic medium utilized in the practice of thepresent invention may merely consist of a test membrane orchromatographic plate and does not necessarily require the use of anabsorbent pad to produce or generate a direction of flow or path ofmigration for a given test sample, as is typically required in prior artassay strips.

Backing

The magnetic chromatography test strip backing 4 can be made of plastic,glass or other suitably rigid material. The backing length can exceedthe length required to support the test membrane and pads, as may bedesired to serve several fimctions. For example, such extended backinglength can provide a handle or it can display information such as barcodes, fluorescent marks, and colored marks which can aid in thecalibration of the individual test strip and multimode photometer, asdiscussed more fully below.

In order to analyze a multiplicity of samples in a single analysis,there is further disclosed herein certain novel assay strips forperforming such function. Referring now to FIG. 5 a, there is shown atop view of a multiplicity of test strips arrayed in parallel rows on acommon backing 4. The backing 4 has a top side and bottom side and canbe in sheet or roll form and is preferably manufactured from an opaqueplastic sheet material of appropriate color, thickness, and rigidity.Each respective test membrane 1 is sufficiently spaced to avoid fluidcontact between adjoining test membranes 1. An absorbent pad 3 ispreferably positioned to be in fluid contact at one end of the testmembrane 1. FIG. 5 b shows a top view of test strips manufactured usinga single common absorbent pad 3 having fluid contact with all testmembranes in a given row. Placement of test membranes 1 and absorbentpads 3 are such that multiple parallel rows of test strips areadvantageously manufactured on a sheet or continuous web of backing 4.Each row of test strips is positioned with adequate spacing such thatindividual test strips for different rows are not in fluid contact witheach other.

In order to identify the presence of a particular analyte, control,calibrator, or combination thereof, these novel methods of the presentinvention deploy a magnetic field at a specific site upon the testmembrane portion of the test strips of the present invention. Suchmagnetic field, which may be generated by any type of magnetic source,such as a permanent magnet or an electromagnet, is selectivelypositioned such that when applied to a portion of the test membrane,magnetic particles present within a given sample that are flowinglaterally across the test membrane will become substantially bound atthe specific site where the magnetic field is applied. In this regard,the applied magnetic field attracts the magnetic particles forming amagnetic barrier that selectively retains magnetic particles, with theanalyte of interest having complexed thereon with appropriate labelsbound thereto, while allowing the remainder of the reaction mixture tocontinue the flow laterally across such barrier or zone. With respect tothose strips depicted in FIGS. 5 a and 5 b, to generate the desiredcapture zones of lines, a magnetic barrier is formed using a barmagnet(s) 20 laminated or placed in close proximity to the bottom sideof backing 4. The bar magnet(s) or magnetized rail(s) 20 is positionedperpendicular to the test membrane(s) 1 in each row and between saidtest membrane(s) 1 fluid receiving and absorbent ends. A reagent zone 2is positioned at the fluid receiving end of each test membrane.

By selectively applying the magnetic field about or upon the test strip,a capture line is magnetically assembled thereon insofar as magneticparticles are substantially immobilized by the magnetic field at aspecific site of sites situated across the test membrane. The remainingreaction mixture components which are not magnetically bound thuscontinue to flow laterally within the test membrane, typically in a pathof migration toward an absorbent pad. Advantageously, such method allowsmore than one analyte, control, calibrant, or combination of these to bequantitatively assayed on a single test strip. Accordingly, it is anobject of this invention to provide a useful method for the performanceof assays, e.g. biological assays.

While the test strips depicted in FIGS. 1 a-1 c and FIGS. 2 a-2 b depictonly one section of test membrane disposed between a reagent pad and anabsorbent pad, it will be recognized by those skilled in the art thatwhen more than one analyte, control, calibrator, or combination thereofare to be assayed within a test solution using a single test strip, acascade of reagent zones or pads can be placed down stream from thefirst applied magnetic field. Several schematic examples of flow teststrip assemblies which can be used with magnetic chromatography aregiven:

Single Assay

reagent zone 1/test membrane//absorbent pad

reagent pad 1//test membrane//absorbent pad

Multiple Assay

reagent zone 1/test membrane/reagent zone 2/test membrane//absorbent pad

reagent pad 1//test membrane//reagent pad 2//test membrane//absorbentpad

Opposing Multiple Assay

reagent zone 1/test membrane//absorbent pad//test membrane/reagent zone2

reagent pad 1//test membrane//absorbent pad//test membrane//reagent pad2

where:

symbol/designates a phase boundary within a single chromatographicmedium; and

symbol//designates a union of two separate mediums (chromatographic andother).

As a consequence, the multiple assay examples given causes test solutionto encounter two groups of magnetic particles. The flow of test solutionis unilateral moving from reagent zone or pad 1 at one end of the teststrip to absorbent pad at the opposite end of the test strip. Magneticbarriers are positioned at each test membrane. The first magneticbarrier is positioned across the test membrane prior to reagent zone orpad 2 while the second magnetic barrier is positioned across the testmembrane prior to the absorbent pad. Reagents from reagent zone or pad 2can be used to analyze additional analytes in the test solution or canbe used to perform calibration or quality control.

The opposing multiple assay example given will allow assay of identicalanalytes from separate test solutions. This is advantageous when acalibrator must be assayed simultaneously with a test sample. The flowof test solution is from each reagent pad or zone toward a single commonabsorbent pad. Magnetic barriers are positioned across each testmembrane. It is also anticipated by the invention that magneticchromatography can be used with other multiple assay test stripconfigurations including rosettes, parallel arrays, and others.

In order to manipulate the width (i.e., surface area) of the captureline formed by the application of a magnetic field to the test strip, ithas been unexpectedly discovered that the width of such capture line maybe selectively controlled depending upon the number of magnets and/ordegree of magnetic force applied to the test membrane. In this regard,it has been discovered that by stacking multiple magnets upon oneanother beneath the test membrane where the captures zone is sought tobe formed, the increased number of magnets applied theretocorrespondingly produces an increase in the width of the capture line.As will be appreciated by those skilled in the art, by utilizing agreater degree of magnetic force, the corresponding capture lineproduced thereby will have a greater surface area which, as aconsequence, can be utilized to determine concentration per unit area.Along these lines, it is contemplated that manipulating the magneticfield to produce a wider or narrower capture line or area may proveextremely beneficial. For example, by manipulating the width or surfacearea of the capture line, a means may thus be provided to facilitate theinspection of individual particles utilizing a microscope. Likewise,such selective manipulation of the capture zone may be used to isolatetarget cells from a population of cells, and thereafter performmicroscopic inspection thereof as may be necessary for a givenapplication.

With respect to the dimensions of such magnets that are preferablyutilized in the practice of the present invention, it is currentlybelieved that bar magnets and/or magnetized rails may be utilized whosewidth is between 0.003 to 3.0 inches, and whose length is between 0.010inches to 100 inches. In this regard, it will be understood that suchmagnets, and in particular magnetized rails, may be sized and configuredto generate any degree of magnetic field necessary to form a desiredcapture line and may be readily determined for a given application byone having ordinary skill in the art.

Referring now to FIGS. 6 a and 6 b, there is shown a third embodiment ofa test strip for performing the assays of the present invention. Unlikethe aforementioned embodiments, however, such test strip is specificallydesigned and configured to form a capture line at the point of contactat which a reaction mixture containing magnetic particles is introducedto the strip. In this regard, and in contrast to the otheraforementioned embodiments, the test strip according to the embodimentdepicted in FIGS. 6 a and 6 b does not require the reaction mixture toflow according to a unilateral pathway extending from the absorbent pad,across the test membrane, and ultimately over the magnetic field bywhich the capture line is ultimately created.

As will be recognized by those skilled in the art, by forming thecapture point at the initial point of contact at which the reactionmixture is contacted with the test strip, there is advantageouslyconserved the amount of analyte sought to be detected through the assaywhich, unlike prior art devices and methods, can diffuse away from thecapture zone of bound receptors or otherwise be restrained from reachingsuch capture zone due to nonspecific binding. As is well-recognized inthe art, a significant portion of the analyte sought to be detectedthrough use of conventional assays can go undetected by virtue of thesequential lateral flow that must occur from when the reaction mixtureis introduced to an assay to the point where such reaction mixture comesinto contact with the bound receptors forming the sought-after captureline.

Referring initially to 6 a, such embodiment comprises an elongatebacking 4 having first and second opposed ends and an intermediateportion. As stated above, such backing may be made of glass, plastic, orother suitably rigid material. Formed upon the opposed ends of thebacking are first and second absorbent pads 3′, 3″ which, as discussedmore fully below, caused the reaction mixture to ultimately flowbilaterally from the point of contact at which the reaction mixture isintroduced to the test strip. Affixed underneath the backing 4 is anelongate magnet 30, which is utilized to create the capture line for usein further analysis. In the alternative, the elongate magnet 30 may beproximately positioned underneath the backing 4 in order to yield aplurality of advantages, such as facilitating microscopic applications.

Formed upon the intermediate portion of the backing 4 between the firstand second absorbent pads 3′, 3″, and centered over the magnet 30 arepreferably provided first vertical flow mesh 32 and second lateral flowmesh 34. As illustrated, the vertical flow mesh 32 is formed on top oflateral flow mesh 34, the latter being in contact with the first andsecond absorbent pads 3′, 3″ on opposed sides thereof. Preferably, aportion of the lateral flow mesh 34 is partially positioned underneaththe first and second absorbent pads 3′, 3″ as shown. In order tointroduce the reaction mixture, there is further preferably provided asample well 36 which is specifically designed and configured tointroduce the reaction mixture directly upon the first vertical mesh 32so that the same thereafter sequentially flows to the lateral flow mesh34 and ultimately to the absorbent pads 3′, 3″ via bilateral flow.

According to a preferred embodiment, the vertical flow mesh 32 is formedof polyester, nylon, glass fiber or any other like material and isoriented to facilitate downward flow therethrough in order to filterlarge debris which may be present in the test solution. The lateral flowmesh 34 is similarly formed from polyester, nylon, glass fiber or anyother like material to allow bilateral flow to the respective absorbentpads 3′, 3″. The lateral flow mesh 34 may also be formed from materialshaving magnetic properties, such as metal screens, metalized polyesterand the like. The sample well 36 is preferably formed from anon-magnetic material such as plastic, and in particular polyvinylchloride, so as to not react with the magnetic particles present withinthe reaction mixture.

As an alternative to providing an arrangement of meshes, such as thecombination of first mesh 32 and second mesh 34, discussed above, it iscontemplated that the backing 4, and more particularly the intermediateportion thereof may be formed to have a texturized surface that isconfigured and oriented to direct the flow of sample flowingthereacross. In this respect, it is contemplated that any texturedsurface capable of causing lateral flow of the test solution across theintermediate portion of backing 4 may be utilized in the practice of thepresent invention, and in particularly as a substitute for the lateralflow mesh 34.

Referring now to FIG. 6 b, there is shown the sequence by which an assaymay be performed using the embodiment depicted in FIG. 6 a. Initially,the reaction mixture is deposited within the sample well 36. Due to bothcapillary action and downward gravitational forces, indicated by theletter A, the reaction mixture is sequentially caused to pass throughvertical flow mesh 32, lateral flow mesh 34 and ultimately to absorbentpads 3′, 3″ via lateral flow depicted by the letter B. However, themagnetic particles having the analyte of interest complexed therewithwill be caused to become substantially bound within the magnetic zonedefined by magnet 30 disposed underneath the backing 4. As such, themagnetic particles become captured at the starting point of the assay,as opposed to at some point removed from where the reaction mixture isinitially introduced, as occurs through most conventional strip assays.In the alternative, the use of textured surfaces formed directly on back4, or attached thereon, may be contemplated. More specifically, anytextured surfaces capable of causing lateral flow of the test solutioncan be utilized as a substitute for the lateral flow mesh 34.

Advantageously, the analyte sought to be detected is conserved and isnot allowed to diffuse or otherwise become bound outside the captureline. As such, such embodiment provides enhanced sensitivity that hasnot heretofor been before available. Along these lines, it iscontemplated that the embodiment depicted in FIGS. 6 a and 6 b areexceptionally advantageous for use in isolating particular types ofcells, such as cancer cells, and other large molecules and biologicalstructures. In this respect, because of the size of such structures,coupled with their limited quantities in a given sample (e.g. one cancercell in fifty billion cells, as can be found in a 10 ml blood sample),such structures have been difficult to isolate and detect in the past.In this regard, due to their size, such structures are physicallyprevented from reaching the capture or target sites provided to detecttheir presence. In such applications, it is found that the nylon mesh,as utilized for the lateral flow mesh 34, is exceptionally advantageousinsofar as the same has been shown to substantially increase lateralflow velocity of the reaction mixture away from the capture zone (i.e.,the magnetic field generated by magnet 30), which thus increasesnon-target cell wash out. As a result, target cells may be retained ingreater concentrations and hence may be more easily detected than priorart techniques.

To further enhance the ability of the test strip embodiment depicted inFIG. 6 a and 6 b to increase cell wash out (i.e., facilitate separationand isolation of the target cells from a reaction mixture), there isprovided an alternative bilateral flow test strip depicted in FIGS. 7 aand 7 b. Referring initially to 7 a, there is shown the same componentsof the test strip depicted in FIGS. 6 a and 6 b. Specifically, there isprovided a backing 4 with first and second absorbent pads 3′, 3″ formedupon the opposed ends thereof. Disposed underneath the backing 4 ismagnet 30, provided to generate the magnetic field necessary to producethe desire capture line. There is further provided a sample well 36 forintroducing the reaction mixture to the test strip, followed by verticalflow mesh 32 and lateral flow mesh 34.

With respect to the latter, however, such lateral flow mesh 34 isdesigned to have a generally diagonal orientation relative the verticalflow mesh 32. As will be appreciated by those skilled in the art, byproviding a diagonal orientation of the lateral flow mesh 34 relativethe vertical flow mesh 32, the reaction mixture flowing therethroughexperiences increased velocity in flowing to the opposed absorbent pads3′, 3″. In this regard, the reaction mixture avoids having to flow in aperpendicular direction per the embodiment depicted in FIGS. 6 a and 6b, and, as such, does not experience the degree of impedance as would areaction mixture encounter via the embodiment depicted in FIGS. 6 a and6 b. As discussed above, as an alternative to utilizing a diagonallyoriented lateral flow mesh 34, the intermediate may be to have atexturized surface that is operative to define a specified pathway andflow velocity of the sample flowing thereacross.

Referring now to FIGS. 8-10, and initially to FIG. 8, there is shown anassay system 100 that is multi-configurable in nature and thus enablesthe same to be used in a wide variety of applications that has notheretofore been available. In this regard, the assay system 100, byrelying on the aforementioned principles and structure, can beselectively fashioned to not only accomplish the aforementionedobjectives of providing greater capture and isolation and conservationof fluid specimens, but also provide a simple, rapid, reliable systemthat permits the efficient handling of small or large numbers ofspecimens. Likewise, such assay system 100 is exceptionally effective inperforming assays related to cell isolation, sorting and interrogation,and the generation of a cell culture as discussed more fully below.

As depicted, the system 100 comprises a housing 102 that defines aninterior chamber 104. The housing 102, which is preferably formed fromany suitable rigid material such as plastic and/or metal and have ablack color to minimize photo degradation of potentially-sensitiveproteins, cells and other cellular components, will be configured to andmounted upon base 110. As discussed more fully below, the housing 102may be configured to either be permanently affixed to base 110 or formedto be detachably fastenable therefrom, typically by either mechanicaland/or adhesive means.

Formed upon the center of housing 102 is sample well 106, which asillustrated has a generally frusto-conical configuration that tapers todefine an exit opening 108. As will be recognized by those skilled inthe art, the sample well 106 provides means for depositing a reactionmixture suspected of containing an analyte of interest that willultimately be captured and isolated as discussed above. To that end, thesample well 106 with opening 108 defined thereby will deposit a reactionmixture upon a target area that may optionally be defined by layer ofmesh 112, the latter being desired to rest upon base 110 and,optionally, become sandwiched between housing 102 and base 110.

To facilitate the bilateral flow of the reaction mixture, the interior104 of housing 102 will define spaces for absorbent members, which maybe materials such as wicks or pads 114, 116 as shown that are disposedon opposed sides of the sample well 106. As discussed above, pads 114,116 will be operative to draw away that portion of the reaction mixture,via lateral flow, that does not contain the magnetic particles that willhave complexed with the analyte of interest. With respect to the latter,the same will be retained at the point of deposition upon the targetarea by virtue of its contact with the magnetic field generated bymagnet 118. As discussed above, the magnetic field may be generated by avariety of means known in the art and may be either integrated as partof base 110, detachably fastenable to base 110, or completely detachedtherefrom. Along these lines, it is contemplated that the target areaupon which the analyte of interest is captured will be approximately 0.6cm² compared with 4 cm² of inspection area as per conventional coverslips.

Referring now to FIG. 9, there is shown an exploded view of an assayconstructed in accordance with the assay system 100 of FIG. 8 that, forreasons that will be discussed, can be selectively constructed toperform an enhanced assay function. As illustrated, in its most basiccomponents the assay will comprise housing 102 having sample well 106and aperture 108 formed thereupon. Absorbent pads 114, 116, disposed onopposed sides of the sample well 106 and confined within the interior ofhousing 102, are provided to draw out a portion of the reaction mixturenot containing the magnetic particles complexed with the analyte ofinterest. As further illustrated, a mesh 112 may be provided upon whichabsorbent pads 114, 116 will rest upon such that the reaction mixturewill flow bilaterally thereacross. Pads 114, 116 may also be retained inposition via a sheet of adhesive, shown as 200 in FIG. 11 that isuseftil in certain applications to secure housing 102 into position.Disposed on the center of the mesh 112 is capture area 120 that isaligned to be directly beneath opening 108 so that the sample that isdeployed through the assay will be conserved and captured at discreetarea of isolation. Capture area 120, as discussed more fully below, maytake a variety of forms, and may include a second layer of mesh or, asdiscussed more fully in connection with FIG. 10, may comprise a novelreceiving gel that may provide an enhanced capture mechanism that may beconfigured to form a variety of biochemical functions.

Base 110 is further provided and serves not only as a structural supportfor the other assay components, but may also be useful in performingfurther processing related to the assay. Along these lines, it iscontemplated that base 110 may take the form of a conventional, standardmicroscope slide or tissue culture vessel of any form including dish,flask, or multi-well (an example of which is the commonly known Petridish).

Along these lines, in those applications where the base 110 is utilizedas a structure for capturing cells, the same may be utilized to directlyinitiate the formation of a cell culture. As will be recognized by thoseskilled in the art, the identification, isolation and eventual growth ofa cell culture via a one step assay process has not heretofore beenavailable, and much less as efficient and effective as those of thepresent invention at not only being able to detect and isolateexceedingly small quantities of cells that may be present in a sample,but thereafter directly grow such cells to generate a cell culture.

Given the foregoing description of the components of the assay system100 discussed in relation to FIGS. 8 and 9, there is now provided adiscussion as to how the various components can be selectively chosenand interconnected to perform specific types of assays having anenhanced function for performing a given assay procedure.

In the embodiment depicted, the assay system 100 is operable and readilyscalable from single slide bench top applications to a continuous highthroughput screening environment. Specifically, the assay can be usedfor a single slide bench top applications, small batch testing,automated batch processing and continuous high throughput screening(HTS). In such applications, the base 110 may take the form of astandard microscope slide, which easily adapts to existing fluidhandling systems, such as Gilson and Tecan Systems. In this regard, suchsystems allow for large batch processing and walk-way automation, andeliminate the need for massive robotic transfer systems and otherexpensive capital equipment.

For procedures involving cell isolation, sorting and interrogation, theassay system 100 can be selectively modified to suit a particularapplication. In applications involving cell engineering andinterrogation, the basic assay system depicted in FIGS. 8 and 9 will bepreferably formed from all non-detachable parts comprised of housing102, absorbent pads 114, 116, mesh 112 and base 110, the latterpreferably taking the form of a microscope slide. In such configuration,the assay will preferably be able to handle samples of 4-8 ml. andfurther allow for serial additions of reagents for immunofluorescentanalysis. Analysis of staining can be performed either manually ordigitally using an inverted microscope usually at 40×.

For cell engineering and expansion applications, the assay can bemodified such that the housing 102 and absorbent pads 114, 116 will beselectively removable to thus leave mesh 112 in place. Suchconfiguration allows for simple culture methods and post-purification oftarget cells (e.g., transfection, drug resistance, etc.). Suchconfiguration also provides a platform for post-culture analysis such asimmunofluorescence, FISH, etc. analysis of culture staining can beperformed either manually or digitally using an inverted microscope,usually at 40×.

For applications involving cell resistance and interrogation, the assaysystem 100 may be configured such that the housing, absorbent pads 114,116 and mesh 112 are removable from base 110. In such applications, atarget area (not shown) will be defined directly upon base 110, uponwhich the purified product will be left naked. Such an assay will beideal for placing cells in altered medium, such as Matrigel, for drugresistance testing. Moreover, such configuration may further provideideal for FISH testing. To that end, the assay according to suchconstruction may preferably support flexible microscope evaluation usingeither inverted or conventional, manual or digital equipment (from10×-100×).

For applications involving cell sorting and serial interrogation, theassay can be configured such that the housing 102 is removable. As aresult, base 110 is left with the absorbent pads 114, 116 remainingattached thereto. The mesh 112 may be selectively fashioned andconfigured to be either left on site upon base 110, or selectivelyremoved therefrom. Such configuration permits serial additions ofreagents for assays such as FISH, immunofluorescence, and cell-basedPCR.

As a further application particularly well adapted for the generation ofa cell culture, there is shown in FIGS. 11-14 a systematic process bywhich the multi-conformable system 100 can be utilized for cellisolation and culture. According to such method, there is providedhousing 102 that may optionally include absorbent pads 114, 116 (notshown). A bottom layer of adhesive 200 will extend across the undersideof housing 102 except for a discrete area 202 surrounding the opening108 through which the sample will pass. Such application will furtherpreferably utilize a culture dish 110′ as the base member that willdefine an interior portion 204 within which the housing 102 will bereceived and secured into position via adhesive layer 200. Along theselines, it is contemplated that an adhesive liner (not shown) will beutilized to form a protective seal about adhesive layer 200 and will beremoved prior to the adhesive attachment of the housing 102 to theinterior surface 204 of culture dish 110′.

Once so positioned, as shown in FIG. 12, a liquid sample 206 will bepoured into the well 106 of housing 102, as discussed above. Tofacilitate the ability of the analyte (i.e., cells) of interest to beisolated at a desired locus, magnet 208 will be deployed in axialalignment with the opening 108 through which the sample 206 isdeposited. As will be readily appreciated, sample 206, prior to beingintroduced into the assay system, will be incubated with magneticparticles that are specific for the target cell type sought to beisolated and cultured.

Once the sample has been deposited within the housing 102, a growthmedium 210 can be deposited through well 106 to thus wash the capturedcells and promote cell growth. Advantageously, by virtue of the factthat the target cells are retained into position via the magneticattraction between the magnetic particle-antibodies specific for suchtarget cells and the magnet 208 positioned below the point of depositionof culture dish 110′, such target cells will be caused to remain inrelatively fixed position. The remaining fluid sample and excess growthmedium, in contrast, will be drawn away from the capture site at whichthe target cells are biased and flow bilaterally to the opposed sides ofthe well 106 and absorbent pads 114, 116, respectively. Along theselines, it has advantageously been discovered that the use of an adhesivelayer 200 will not interfere with the fluid dynamics associated with theassay.

Following the administration of a growth medium to wash the capturedcells, the housing 102 can be removed from floor 204 of the culture dish110′, as shown in FIG. 14. Advantageously, the target cells of interestwill be isolated at the point of deposition and thus may be allowed togrow to produce a, desired cell culture. To facilitate that end, it willbe understood by those skilled in the art that culture dish 110′ may betreated with any of a variety of substances to facilitate and promotethe formation of a cell culture. Additionally, it will be understoodthat the captured cells 212 may be viewed microscopically or examinedper any of a variety of conventional modalities.

Referring now to FIGS. 15-19, and initially to FIGS. 15 and 16, there isshown enhanced systems through which isolation of a target analyte, andin particular a specific cell type, can be more easily and readilyisolated from a fluid sample. Referring initially to FIG. 5, such systemis encompassed within a texturized surface 220 formed on the undersideof housing 102 and situated within the area 202 formed within adhesivelayer 200. The texturized area 220 is preferably situated about opening108 through which the fluid sample containing the target cell type willpass. Such texturized surface 220, shown more clearly in FIG. 16, isprovided with a plurality of objects or structures 222, which may takethe form of cylinders, pillars, frusto-conical protuberances, moundsand/or one or more channels extending thereabout that are operative torestrict or impede the flow of the sample passing through aperture 108and ultimately to absorbent pads 114, 116 (not shown) held behindadhesive layer 200. In this respect, such texturized surface 220 withstructures 222 formed thereon are operative to divert or otherwiseincrease resistance of the sample flow from opening 108 to the absorbentpads such that the rate of flow occurs at a slower rate and/or with lesspressure.

As it will be readily appreciated by those skilled in the art, the fluidsample deposited within well 106 (not shown) and ultimately throughopening 108 and to absorbent pads 114, 116 (not shown) may in someapplications flow with sufficient velocity such that the target cell ofinterest, despite being complexed with a magnetic-particle antibody,will cause the target cell to flow past the site at which the magneticfield is deployed, thus resulting in a loss of the target cell. As willbe appreciated, because the target cell type may be either exceptionallyfragile and/or present in very limited quantities, it is often timesessential that the capture of such cell be made as sensitive aspossible.

The texturized surface 220, via structures 222 formed thereon,accomplishes this end by diverting the flow of the sample in indirectpathways, increases the time that the target cell types with magneticparticle-antibodies bound thereto will be attracted by and drawn to themagnetic force applied at the specific locus. In this regard, the targetcell types having the magnetic particle-antibody bound thereto will bedrawn to the magnetic force that is specific only for those particles,thus allowing the remaining fluid sample undesirable cells, analytes,dispersed therein, to freely flow away from the magnetic field and tothe absorbent pads 114, 116.

Likewise, the structures 222 formed upon texturized surface 220 alsopresent a physical barrier that increases collisions, blockages and flowdiversions of a magnitude that prevents the cell type of interest tofreely flow past a field of magnetically-attractive forces that oftenwould cause the target cells to remain at the desired locus.

As will be readily appreciated by those skilled in the art, thetexturized surface 220 may be formed to have a variety ofconfigurations, and may include any of a variety of uniform ornon-uniform distribution of structures, channels and the like, all ofwhich being operative to impede the flow of the fluid sample to thusenable the target cell line of interest to be more readily captured atthe site of deposition through aperture 108. Along these lines, it willbe further appreciated that area 202 formed upon adhesive layer 200 maybe selectively sized to suit a particular application.

Referring now to FIGS. 17-19, and initially to FIG. 17, there is shown afurther means by which a sample flow passing from aperture 108 of thehousing 102 (not shown) across to absorbent pads 114, 116 (not shown)can be achieved to enhance the ability of the assay system 100 to moreprecisely capture target cells of interest. As illustrated, there isprovided a specialized texturized surface 230, which is formed upon base110, as opposed to the underside of housing 102. The texturized surface230, as more clearly seen in FIG. 18, is provided with a variety ofstructures 232, similar to that discussed above in connection withtexturized surface 220, that are operative to impede or restrict theflow of the fluid sample passing thereacross to thus enable target cellssought to be isolated from flowing across the magnetic locus where thecells are sought to be retained and on to absorbent pads 114, 116 (notshown). As illustrated in FIG. 18, the structures 232 may be arranged ina uniform pattern of pillars or cylindrical members to thus define aplurality of physical barriers around which the fluid sample must flow.In one embodiment, the structures will have a height of approximately 20microns and space apart by approximately 50 microns. As will beappreciated, however, a variety of alternative structures, channels, andcombinations thereof, whether arranged in a uniform or non-uniformmanner, may be deployed to effectuate the impedance necessary torestrict the flow of the sample to thus facilitate capture of the targetcells of interest. Accordingly, it should be recognized that thetexturized surfaces, whether discussed in the context of a texturizedsurface formed upon housing 102, as discussed in regard to FIG. 17 and16, or the second texturized surface discussed in connection with base110, of FIGS. 17 and 18, should be construed as broadly as possible.

Referring now to FIG. 19, there is shown a cross-sectional view of FIG.18. As illustrated, pillars 232 define rows of physical barriers aroundwhich a fluid sample must flow, thus impeding flow and creating physicalbarriers that reduce the ability of objects suspended within the sample,and in particular the target cells of interest, from flowing thereacrossand onto absorbent pads 114, 116 (not shown) which would result in theloss of such cells. By virtue of the same mechanisms discussed above,the impedance or restriction of flow increases the time and ability thatthe target cell types having the magnetic particle-antibodies boundthereto to become retained by the magnetic force applied thereto andthus remain captured, as opposed to passing through and being lost.

In yet a funrther refinement depicted in FIG. 10, it is contemplatedthat the target area may optionally be defined by a gel 120 that isoperative to facilitate the capture, isolation and possible furtherbiochemical processing of the target analyte captured thereat. Such gel,which can comprise any of a variety of biocompatible gels well-known tothose in the art, such as polyacrylamide gels utilized inelectrophoresis and the like, may serve as a medium for capturing andisolating cells, intracellular structures, proteins, genes, and thelike. Such gel 120 may be either formed on a mesh backing 112 as shown,or otherwise formed directly on base 110. Other substrates (not shown)may also be utilized to support gel 120 at or around the target area. Toenhance the capability of the gel 120 to capture and isolate cells,intracellular structures, etc., it is contemplated that such gel may beprovided with receptors 122 specific for the target analyte of interest.In this regard, by suspending such receptors 122 or, alternatively,having the receptors 122 bound to a solid phase, can facilitate theability of the assays of the present invention to provide enhancedcapture and isolation of target analytes.

In addition to or separate from such receptors 122, the gel, as depictedas 120 in FIG. 10, may be provided with other chemical modifiers,reagents or enzymes, depicted as 124, that may be operative to providefurther biochemical processing of the analyte of interest. Onecontemplated application includes the use of digestive enzymes toselectively cleave proteins or otherwise digest target intercellularcomponents for further isolation and identification. To that end, it iscontemplated that formulation and composition of such gel compositionshaving the desired reagents, receptors, enzymes, and the like will bereadily known and capable of being made by those skilled in the artusing known techniques. In a further contemplated application, the gel120 may serve as a growth medium for cells to thus enable a target cellof interest to be expanded or propagated to thus create the formation ofa cell culture. To attain such objective, it is contemplated that avariety of nutrients and other growth media well-known to those skilledin the art may be incorporated as part of gel 120.

Advantageously, the assays of the present invention offer greaterflexibility than microtiter plates and can be efficiently used forcellular sub-population analytic assays while simultaneously determiningthe presence of aberrant DNA, mRNA transcription expression, and proteintranslation expression (simultaneous FISH/IF). The assays of the presentinvention can likewise be used to isolate and sort cells, proteins orgenes with one self-contained unit, thereby eliminating the need forseparation columns. Moreover, by virtue of the direct addition ofsample, reagents, and cell wash solutions through the well 106eliminates cell transfer steps, really expedites the assay procedure,enhances cell capture, lowers cell loss, and minimizes the chance oferror. Along these lines, it is further contemplated that the assays ofthe present invention can be specifically configured such that theinterconnection between housing 102 and base 110 will define one or moreopenings that will define a channel through which further post-assayassessment and manipulation can be conducted. For example, it iscontemplated that the interconnection between base 102 and 110, whetheror not such structures are permanently affixed or detachable from oneanother, such elements 102, 110 may cooperate to define one or morechannels through which cellular testing may be conducted or otherwiseallow reagents to be introduced therethrough to facilitate cellularlysing/digestion and/or other reagents to facilitate separation andisolation of target proteins, organelles, and the like.

As will be appreciated by those skilled in the art, the assays of thepresent invention, by virtue of their multi-functional configurationsmay be used in a number of applications, particularly with respect foruse in drug discovery. To that end, the assays of the present inventionmay be exceptionally effective to determine the effect of the compoundon a cell for efficacy and potential toxicity. As is well-known, currenttechniques utilized to differentiate the expression profiles of purecell populations, advanced separation techniques and novel geneexpression methods are complex, costly, labor intensive, and not readilyscalable. Not only do the assays of the present invention overcome suchdisadvantages of the prior art, the same actually have far more widespread application. Along these lines, the assays of the presentinvention are exceptionally effective at forming both targetidentification and target validation procedures. With respect to theformer, which include procedures such as cell isolation, organelle orsorting, and protein fractionation, and the latter, which can involvespecific protein isolation, immune assays, and molecular analysis, oftentimes required capture and isolation of cells, proteins or genes thatremain functionally and structurally intact. At times, however, suchtargets are often fragile, membrane bound, or of exceptionally small orlarge size. Despite such difficult properties or characteristics,however the assays of the present invention are well-suited tofacilitate the separation and isolation of such targets in a manner thatis superior to that of the prior art.

The present invention further includes a novel analyzer having amultimode photometer module included therein which can measure frontsurface fluorescence (fluorimetry mode), luminescence (luminometrymode), and reflectance (densitometry mode) at a single focal point on atest strip. The use of multiple optical methods at a single focal pointprovides information regarding the quality and structure of anindividual capture line as well as the amount of analyte, control, orcalibrator present at the capture line. Thus an object of the inventionis to minimize accuracy and precision problems associated with teststrips by interrogating important test strip locations using two or moreoptical methods.

As illustrated in FIG. 3 a, the multimode photometer consists of anoptical canopy 9 a and a base 9 b which cooperate to form an opticaltunnel 9. The optical tunnel 9 aligns light sources and photodetectors,with magnetic sources and test membranes, chromatographic plates, etc.to form optical paths. In this regard, base 9 b includes a channelformed therein for receiving a test strip of the aforementioned variety.The base 9 b further preferably includes a magnetic source fixed thereinor fixed relative the channel to thus create the desired capture line ata specified location within the optical tunnel 9. For example, amagnetic source, such as a magnet, may be placed beneath the base of theoptical tunnel 9 b such that the test strip rests in the channelsituated thereabove.

The optical canopy 9 a is formed to have a ceiling through which a lightsource may be transmitted, and angled sidewalls through which theresultant reflected light may be emitted. As will be recognized by thoseskilled in the art, the multimode photometer, and more particularly theoptical tunnel defined thereby, may be extruded, machined, or moldedfrom any of a variety of suitable opaque materials, including but notlimited to PVC, ABS, or anodized aluminum. As such, the optical tunnel 9of the present invention may be fabricated inexpensively frominexpensive materials.

Referring now to FIG. 3 b there is schematically illustrated thecomponents utilized for analyzing a test strip with the multimodephotometer of the present invention. Initially, an excitation path 6 isformed from the light source 7 to a focal point 8 at the base 9 b of theoptical tunnel 9. As will be readily appreciated, the magnetic sourceincorporated into the base 9 b for forming the capture line on a giventest membrane or chromatographic plate will be precisely aligned withthe excitation path 6 such that the path 6 is directly aimed at thecapture line produced by such magnetic source. As will be appreciated,light emitting diodes (LEDs), laser diodes, mercury vapor lamps, andxenon lamps are among many suitable light sources which can be used. Ifnecessary, an optical filter 10 can be used to select an excitationwavelength 6. This excitation filter 10 can be placed on either side ofthe canopy wall 9 a provided, however, the same is in the excitationpath 6 between the light source and test strip 11. When a test strip 11is inserted into the optical tunnel 9, such strip is held in position atthe base and intersects the excitation path 6 at the focal point 8.

Emission paths 12 are formed from the focal point to one or morephotodetectors 13. Apertures are positioned using a radial geometry inthe canopy wall 9 a at angles which optically align each photodetector13 with the focal point 8. Light pipes, optical fibers, and other waveguides can be used to transmit emission light to the photodetectors 13.Excitation light 6 excites fluorophores present on the test strip 11 atthe focal point 8, which then emit light 12 of a longer wavelength. Ifluminescence is used excitation light 6 is not required and can beomitted during luminescence measurement. Emission filters 14 are used tospecifically select the emission wavelength 12 of the light emitted fromthe flourescent or luminescence and to remove traces of excitation light6. As will be appreciated by those skilled in the art, such emissionfilter 14 can be placed on either side of the canopy wall 9 a providingit is in the emission path 12 between the photodetector 13 and teststrip 11.

Reflectance paths 15 are also formed from the focal point 8 to one ormore photodetectors 16. Such reflectance path 15 carries both excitation6 and emission light 12. If necessary, excitation filters 10 can be usedto specifically select the excitation wavelength 6 of the lightreflected from the test strip and to revoke traces of emission light 12.This excitation filter 10 can be placed on either side of the canopywall 9 a providing it is in the reflectance path 15 between thephotodetector 16 and test strip 11.

The filters 10 and 14 can be of the type known in the art asinterference filters, due to the way in which the same block out-of-bandtransmissions. In this respect, interference filters exhibit anextremely low transmission outside of their characteristic bandpass and,as such, are very efficient in selecting the desired excitation andemission wavelengths.

As will further be appreciated by those skilled in the art, an opticaltunnel can have multiple focal points at which photometric measurementscan be made simultaneously, which advantageously allows multiple pointson a test strip to be used for sample analysis and/or calibration. Insuch applications, optical components, such as LEDs, photodiodes, andinterference filters, may be clustered at each focal point along theoptical tunnel.

As prospectively illustrated in FIGS. 4 a and 4 b, there is showndifferent views of an optical tunnel equipped with two optical clustersas may be utilized for multispectral analysis. A light source 7 (LEDs 7a and 7 b are shown) is positioned above an excitation filter 10(filters 10 a and 10 b are shown) which in turn covers each excitationaperture (not shown). Two of four photodiodes 13 a, 16 a with filters10, 14, as shown in the cross-sectional view of FIG. 4C, are mounted onthe canopy 9 a. A bar magnet 20 a, as shown in FIG. 4C, is positioned atthe base of the optical tunnel beneath each focal point 8 such thatappropriate spectrophotometric analysis may be made at each location.

Although believed to be apparent from the foregoing discussion, there isprovided herebelow a variety of examples by which the novel magneticassays and methods of the present invention may be utilized in a varietyof applications. As will be appreciated by those skilled in the art, forthe purpose of discussion in the following examples the term “testsolution” can mean test sample, test calibrator, or test controlmaterial.

EXAMPLE 1

A test strip is manufactured according to the description given inFIG. 1. The backing 4 is extended in length beyond the absorbent pad 3end to allow application of bar codes, fluorescent markings, and otherindicators to the backing 4. Reagent zone 2 contains streptavidinconjugated magnetic particles, buffers, stabilizers, surfactants, andother reagents in dry form.

The test strip 11 is inserted absorbent pad 3 end first into the opticaltunnel 9. Indicators on the test strip are interpreted as calibrationinformation by the analyzer. For example, the analyzer verifies that thesame bar code was read at both focal points 8 a and 8 b and storesreflectance and fluorescence values for photodetectors 13 and 16. Thecalibration information and measured values are used by the analyzer toverify the quality and structure of an individual capture line as wellas the amount of analyte, control, or calibrator present at the captureline, and to verify the performance of each optical module.

In a separate container the operator adds a measured volume of sample toa measured volume of test reagents and mixes them to form a reactionmixture. The test reagents include biotin conjugated anti-beta HCG, andfluorescent microsphere conjugated anti-alpha HCG which cooperativelybind HCG molecules present in the sample.

A measured volume of this reaction mixture is applied to the test stripreagent zone 2 it forms a new reaction mixture which contains magneticparticles in suspension as buffers, stabilizers, surfactants, and otherreagents previously dried on the reagent zone 2. The magnetic particlesbind the biotin conjugate in all of its complexed forms including thosewhich have formed a cooperative complex (sandwich assay) with HCG andthe anti-alpha HCG conjugate. Thus, fluorescent microspheres areindirectly bound to magnetic particles in proportion to the amount ofanalyte present in the reaction mixture.

As the magnetic particles suspended in the reaction mixture flowlaterally within the plane of the test strip 11 they encounter amagnetic field applied using a bar magnet 20 attached to the base 9 b ofthe optical tunnel 9. The applied magnetic field attracts the magneticparticles forming a magnetic barrier that selectively retains themagnetic particles at the focal point 8 while allowing reaction mixtureto continue to flow laterally across this barrier toward the absorbentpad 3.

A measured volume of wash solution can also be added subsequent to theaddition of reaction mixture. This will reduce the amount of fluorescentmicrospheres retained by the test membrane 1 and magnetic particles dueto non-specific binding.

The analyzer monitors and compares photodetectors 16 a and 16 bmeasuring reflectance at the focal point 8 a and 8 b. The reflectedlight intensity at the focal point 8 a decreases as the magneticparticles are retained by the magnet 20. The reflected light intensityat focal point 8 b is a background (blank) measurement used to correctfor differences between individual test strips and sample matrixeffects. This allows the analyzer to determine whether the magneticparticles have been properly captured at focal point 8 a, and to rejectsamples which are hemolyzed or contain elevated amounts of chromophoressuch as bilirubin. If the reflected light intensity is not withinspecification at focal points 8 a and 8 b during a predefined elapsedtime the test is determined invalid and no result is reported.

Alternating with photodetectors 16 a and 16 b, the analyzer alsomonitors and compares photodetectors 13 a and 13 b measuringfluorescence. The emitted light intensity at focal point 8 b is abackground (blank) measurement used to correct for non-specific binding,differences between individual test strips, and sample matrix effects.The analyzer compares the blank emission measurement at 8 b and testemission measurement at 8 a and calculates HCG concentration.

EXAMPLE 2

Example 2 mirrors Example 1 but for the following differences:

Reagent zone 2 contains all test reagents prepackaged in unit dose driedform including: streptavidin conjugated magnetic particles, biotinconjugated anti-beta HCG, and fluorescent microsphere conjugatedanti-alpha HCG which cooperatively bind HCG molecules present in thesample. Reagent zone 2 also contains buffers, stabilizers, surfactants,and other reagents in dry form.

The operator adds a measured volume of test solution directly to reagentzone 2.

EXAMPLE 3

Example 3 mirrors Example 2 but for the following differences:

Anti-alpha HCG is conjugated using alkaline phosphatase, instead offluorescent microspheres.

A measured volume of fluorescent substrate is added to the reagent zone2 subsequent to the addition of a measured volume of wash solution.

EXAMPLE 4

Example 4 mirrors all of the above examples but for the followingdifferences:

Example 4 substitutes a reagent pad 5 for reagent zone 2 in each of thepreceding examples.

EXAMPLE 5

A test strip is manufactured according to the prescription given inFIG. 1. The backing 4 is extended in length beyond the absorbent pad 3end to allow application of bar codes, fluorescent markings, and otherindicators to the backing 4. Reagent zone 2 contains streptavidinconjugated magnetic particles, buffers, stabilizers, surfactants, andother reagents in dry form.

In a separate container, the operator adds a measured volume of testsolution (containing cells, cell lysate, total RNA) to a measured volumeof test reagents and mixes them to form a reaction mixture. The testreagents include biotinylated oligo (dT) probe and a 5′ fluorescent dyelabeled DNA hybridization probe specific for Chlamydia.

A measured volume of this reaction mixture is applied to the test stripreagent zone 2. As the reaction mixture comes in contact with thereagent zone 2 it forms a new reaction mixture which contains magneticparticles in suspension as well as buffers, stabilizers, surfactants,and other reagents previously dried in the reagent zone 2. thebiotinylated oligo (dT) probe hybridizes specifically to the 3′ poly(A)region of all mRNA present in the test solution. Consequentially, allmRNA is bound to the magnetic particles via a biotin/streptavidin bond.Labeled hybridization probe, in contrast, binds only target mRNA. Themagnetic particles bind the biotinylated oligo (dT) probe in all of itscomplexed forms including those which have formed a cooperative complex(hybrid) with Chlamydia mRNA and the fluorescent dye labeled DNAhybridization probe specific for Chlamydia. Thus, fluorescent dye isindirectly bound to magnetic particles in proportion to the amount ofChlamydia mRNA present in the reaction mixture.

As the magnetic particles suspended in the reaction mixture flowlaterally within the plane of the test strip 11 they encounter amagnetic field applied using a bar magnet 20 attached to the base 9 b ofthe optical tunnel 9. The applied magnetic field attracts the magneticparticles forming a magnetic barrier that selectively retains themagnetic particles at the focal point 8 while allowing reaction mixtureto continue to flow laterally across this barrier toward the absorbentpad 3.

A measured volume of wash solution can also be added subsequent to theaddition of reaction mixture. This will reduce the amount of labeled DNAprobe retained by the test membrane 1 and magnetic particles due tonon-specific binding.

The analyzer monitors and compares photodetectors 16 a and 16 bmeasuring reflectance at the focal point 8 a and 8 b. The reflectedlight intensity at the focal point 8 a decreases as the magneticparticles are retained by the magnet 20. The reflected light intensityat focal point 8 b is a background (blank) measurement used to correctfor differences between individual test strips and sample matrixeffects. This allows the analyzer to determine whether the magneticparticles have been properly captured at focal point 8 a, and to rejectsamples which are hemolyzed or contain elevated amounts of chromophoressuch as bilirubin. If the reflected light intensity is not withinspecification at focal points 8 a and 8 b during a predefined elapsedtime the test is determined invalid and no result is reported.Alternating with photodetectors 16 a and 16 b, the analyzer alsomonitors and compares photodetectors 13 a and 13 b measuringfluorescence. The emitted light intensity at focal point 8 b is abackground (blank) measurement used to correct for non-specific binding,differences between individual test strips, and sample matrix effects.The analyzer compares the blank emission measurement at 8 b and testemission measurement at 8 a and calculates Chlamydia concentration ordetermines simply if Chlamydia is present in the test solution.

EXAMPLE 6

Other detection methods can be used with magnetic chromatography. Inthis example, x-ray film is used to detect the presence of target DNA ina population of transfected cells. PCR amplification of CDNA present ineach test solution is accomplished using P32 labeled nucleotides.Amplified DNA is hybridized using 5′ biotin DNA hybridization probeforming a reaction mixture which is applied to the test strip reagentzone 2 containing streptavidin conjugated magnetic particles.

Utilizing a test strip of the variety depicted in FIG. 5 b, a washsolution is applied to reagent zone 2 subsequent to application of thereaction mixture.

A sheet of x-ray film is placed on top of said test strip array andexposed for a suitable length of time.

A visible band is seen on the developed x-ray film whose positioncorresponds with a sample which has tested positive for the target DNA.

EXAMPLE 7

Example 7 mirrors Example 6 but for the following differences:

Said PCR amplification is accomplished using 5′ fluorescent dye labeledprimer.

Said test strip array is positioned within a fluorescent scanner.

Said fluorescent scanner detects a fluorescent band whose positioncorresponds with a sample testing positive for the target DNA.

EXAMPLE 8

Example 8 mirrors Example 1 but for the following differences:

Said backing 4 is a microscope slide.

Said magnet 20 is positioned above said test membrane 1, so that magnet20 is not in contact with test membrane 1.

A fluorescent microscope is used to count individual fluorescentmicrospheres bound to magnetic particles.

EXAMPLE 9

A test strip is manufactured according to the description given inFIG. 1. The backing 4 is extended in length beyond the absorbent pad 3end to allow application of bar codes, fluorescent markings, and otherindicators to the backing 4. Reagent zone 2 contains streptavidinconjugated 0.86 micron magnetic particles, anti-mouse IgG conjugated 150nm magnetic particles, buffers, stabilizers, surfactants, and otherreagents in dry form.

The test strip 11 is inserted absorbent pad 3 end first into the opticaltunnel 9. Indicators on the test strip are interpreted as calibrationinformation by the analyzer. For example, the analyzer verifies that thesame bar code was read at both focal points 8 a and 8 b and storesreflectance and fluorescence values for photodetectors 13 and 16. Thecalibration information and measured values are used by the analyzer toverify the quality and structure of an individual capture line as wellas the amount of analyte, control, or calibrator present at the captureline, and to verify the performance of each optical module.

In a separate container the operator adds a measured volume of sample toa measured volume of test reagents and mixes them to form a reactionmixture. The test reagents include biotin conjugated goat anti-beta FSH,and fluorescent microsphere conjugated goat anti-alpha FSH whichcooperatively bind FSH molecules present in the sample. The testreagents also include mouse anti-beta LH, and fluorescent microsphereconjugated goat anti-alpha LH which cooperatively bind FSH moleculespresent in the sample.

A measured volume of this reaction mixture is applied to the test stripreagent zone 2. As the reaction mixture comes in contact with thereagent zone 2 it forms a new reaction mixture which contains 0.86micron and 150 nm magnetic particles in suspension as well as buffers,stabilizers, surfactants, and other reagents previously dried on thereagent zone 2. The 0.86 micron magnetic particles bind the biotinconjugate in all of its complexed forms including those which haveformed a cooperative complex (sandwich assay) with FSH and theanti-alpha FSH conjugate. Thus fluorescent microspheres are indirectlybound to magnetic particles in proportion to the amount of analytepresent in the reaction mixture. The 150 nm magnetic particles bind themouse anti-beta LH conjugate in all of its complexed forms includingthose which have formed a cooperative complex (sandwich assay) with LHand the goat anti-alpha LH conjugate. Thus, fluorescent microspheres areindirectly bound to magnetic particles in proportion to the amount ofanalyte present in the reaction mixture.

As the magnetic particles suspended in the reaction mixture flowlaterally within the plane of the test strip 11 they encounter a firstmagnetic field applied using a bar magnet 20 a attached to the base 9 bof the optical tunnel 9. The applied magnetic field is of sufficientstrength that it provides a magnetic barrier that selectively retainsthe 0.86 micron magnetic particles at the focal point 8 a while allowingreaction mixture including 150 nm magnetic particles in suspension tocontinue to flow lateral across this barrier toward the absorbent pad 3.

As the 150 nm magnetic particles suspended in the reaction mixture flowlaterally within the plane of the test strip 11 they encounter a secondmagnetic field applied using a second bar magnet 20 b (not shown)attached to the base 9 b of the optical tunnel 9. The second appliedmagnetic field is significantly stronger than said first appliedmagnetic field. This second applied magnetic field provides a magneticbarrier that selectively retains the 150 nm magnetic particles at thefocal point 8 b while allowing reaction mixture to continue to flowlaterally across this second magnetic barrier toward the absorbent pad3.

A measured volume of wash solution can also be added subsequent to theaddition of reaction mixture. This will reduce the amount of fluorescentmicrospheres retained by the test membrane 1 and magnetic particles dueto non-specific binding.

The analyzer monitors and compares photodetectors 16 a and 16 bmeasuring reflectance at the focal point 8 a and 8 b. The reflectedlight intensity at the focal point 8 a decreases as the magneticparticles are retained by the first magnet 20 a and second magnet (notshown). The reflected light intensity at focal points 8 a and 8 b aremeasurements used to determine whether the magnetic particles have beenproperly captured at focal points 8 a and 8 b. If the reflected lightintensity is not within specification at focal point 8 a and 8 b duringa predefined elapsed time the test is determined invalid and no resultis reported.

Alternating with photodetectors 16 a and 16 b, the analyzer alsomonitors and compares photodetectors 13 a and 13 b measuringfluorescence. The emitted light intensity at focal points 8 a and 8 bare used to calculate FSH and LH concentrations respectively. Theanalyzer compares these emitted light intensities with those of testsolutions containing known concentrations of FSH and LH, based upon suchparameters, and calculates FSH and LH concentrations. It is to befurther understood that various additions, deletions, modifications andalterations may be made to the above-described embodiments withoutdeparting from the intended spirit and scope of the present invention.In this regard, it should expressly be recognized that in addition tothe magnetically-generated capture lines formed herein, additionalcapture lines may be formed as per conventional test strip assays whichincorporate the use of bound receptors formed upon a test membrane.

EXAMPLE 10

A test strip is manufactured according to the description given in FIGS.7 a and 7 b. The backing 4 is formed from a microscope slide. Thelateral flow mesh 34 (chromatographic medium or stationary phase)consists of a 105 micron pore size mesh constructed from woven nylonfiber. The edges of the respective mesh 32, 34 are adhered to themicroscope slide backing 4 using any suitable adhesive tape. Thiscreates an adhesive free zone and allows the lateral flow mesh 34 tomake direct contact with the microscope slide backing 4. The well 36 ismounted concentric with the respective mesh 32, 34 using any suitableadhesive or any other mechanical means which does not interfere withbilateral flow B of the test solutions. The absorbent pads 3′, 3″ are ofapproximately equal dimensions having a total absorbent capacity greaterthan the combined volumes of liquid reagents, test solutions, washsolutions and the like. As illustrated in FIGS. 7 a and 7 b, bothabsorbent pads 3′, 3″ are in fluid contact with the lateral flow mesh34.

In a separate container, the operator may add a measured volume of testsolution (e.g., peripheral blood or bone marrow suspected of containingcancer cells) to a measured volume of test reagents and mixes them toform a reaction mixture (mobile phase). If desired, common laboratoryprocedures may be used to remove the red blood cells from the testsolution prior to mixing with the test reagents. The test reagents maycomprise magnetic particles conjugated using antibodies specific for atleast one human cancer cell type. These antibodies bind to the targetedcancer cell type(s) to make them magnetic. Subsequently, a measuredvolume of fluorescent dye labeled antibodies specific for the samecancer cell type(s) are added to the same reaction mixture. Theseantibodies may bind to the remaining available sites on the cancer celltype(s) to make them fluorescent. Therefore, the reaction mixturecontains cancer cell type(s) that have been rendered both magnetic andfluorescent.

The test strip is strategically placed above the bar magnet 30 such thatan opening at the bottom of the well 36 is positioned above the barmagnet 30. In one embodiment of the present invention, the opening atthe bottom of the well 36 is circumscribed entirely by the bar magnet30. The reaction mixture is deposited into the well 36. Gravitysubsequently causes the reaction mixture to descend through the well 36along direction A until it comes in contact with the vertical mesh 32.Upon contacting the vertical mesh 32, the reaction mixture is forcedthrough the pores within the vertical mesh 32 by a combination ofcapillary action and gravitational force. The reaction mixture may thencontact the lateral flow mesh 34 which conveys the liquid reactionmixture in bilateral directions B′, B″ by the capillary action untilsaid reaction mixture is absorbed by the absorbent pads 3′, 3″.

As the magnetic particles and magnetically labeled cells in the reactionmixture flow from the well 36 to the lateral flow mesh 34, theyencounter a magnetic field that is applied via the bar magnet 30. Theapplied magnetic field forms a magnetic barrier that selectively retainsa majority of the magnetic particles and magnetically labeled cancercells within a narrow capture zone. The non-magnetic remainder of thereaction mixture may continue to flow bilaterally B across this barrierto the absorbent pads 3′, 3″.

Subsequent to the addition of the reaction mixture, a volume of washsolution may be deposited in the well 36 while the test strip remains inposition over the bar magnet 30. The magnetically labeled target cellsand magnetic particles will remain held by the magnetic barrier whilethe non-target cells and unbound fluorescent antibodies are washed fromthe capture zone.

By utilizing the method above, over 100 million cells may be applied toa single microscope slide. Such method reduces the number of images thatmust be generated and examined from 48,000 to less than 40. Those ofordinary skill in the art will immediately recognize the advantage ofcapturing the cells directly on the slide, such that no cells may belost during a transfer step which is known to be a crucial defect ofprior art cell examination techniques.

EXAMPLE 11 Example 11 mirrors Example 10 but for the followingdifferences:

A test strip is manufactured according to the description given in FIG.7. The backing 4 is formed from a microscope slide that has beenfabricated using an optically clear plastic such as polycarbonate oracrylic. The chromatographic medium or stationary phase 34 consists of atexture that is formed directly on the top surface of the microscopeslide during the fabrication process (e.g.; injection molding, hotstamping, or casting). The texture can take any form suitable to achievelateral flow of test solutions and reagents by capillary force.

It is to be understood and appreciated that various additions,deletions, modifications and alterations may be made to theabove-described embodiments without departing from the intended spiritand scope of the present invention. In this respect, while variouspreferred embodiments of the present invention have been illustrated bymeans of specific examples, particularly with respect tomagnetically-generated capture lines and techniques, is to be understoodthat the present invention is in no way to be deemed limited thereto.Accordingly, it is intended that all additions, deletions, modificationsand alterations be included within the scope of the following claims.

1. A multi-conformable assay system for use in performing a magneticchromatography method to isolate at least one target cell present in afluid sample, the assay comprising: a. a housing positionable upon abase, said housing having a sample well formed thereon for receiving areaction mixture having a quantity of magnetic particles-antibodiessuspended therein, said magnetic particle antibodies being operative tobind with said at least one target cell; and b. at least one absorbentmember positionable within said housing and adjacent to said sample wellto absorb a portion of said reaction mixture deposited in said samplewell not containing said magnetic particles.
 2. The assay of claim 1wherein said housing comprises a generally block-like structure definingan upper platform surface, said sample well being formed in the generalmidsection of said upper platform surface, said housing further definingfirst and second voids on opposed sides of said sample well; said systemfurther comprising first and second absorbent pads, said first andsecond absorbent pads being received into dedicated ones of saidcavities of opposed sides of said sample well.
 3. The assay of claim 2wherein said housing is fabricated from a rigid, black material selectedfrom the group consisting of plastic and metal.
 4. The assay of claim 1wherein said base comprises a microscope slide or a cell culture dish.5. The assay of claim 1 wherein said assay further comprises atexturized portion formed underneath said housing about said samplewell, said texturized portion being operative to restrict the flow ofsaid sample flowing from said sample well to said at least one absorbentmember.
 6. The assay of claim 1 wherein said base comprises a microscopeslide and said microscope slide has formed thereon a texturized surfacepositionable below said sample well, said texturized surface beingoperative to restrict the flow of said sample flowing from said samplewell to said at least one absorbent member.
 7. The assay of claim 1wherein said base comprises a cell culture dish and said microscopeslide has formed thereon a texturized surface positionable below saidsample well, said texturized surface being operative to restrict theflow of said sample flowing from said sample well to said at least oneabsorbent member.
 8. The assay of claim 6 wherein said texturizedsurface comprises a plurality of structures uniformly formed upon saidtexturized surface, said plurality of structures being operative toimpede sample flow.
 9. The assay of claim 7 wherein said texturizedsurface comprises a plurality of structures uniformly formed upon saidtexturized surface, said plurality of structures being operative toimpede sample flow.
 10. The assay of claim 9 wherein said texturizedsurface comprises cylindrical pillars.
 11. The assay of claim 4 whereinsaid housing is removable from said base.